A mouse macrophage cell line, P388D1, has been utilized as a model system for 1) production and characterization of thymocyte proliferation factors (TPF) and 2) analysis of the role of T lymphocytes in the regulation of TPF production. High levels of a thymocyte proliferation factor(s) (TPF) were produced by the mouse macrophage cell line, P388D1, when these cells were incubated with activated mouse or guinea pig T lymphocytes, supernatants derived from T lymphocyte cultures, LPS or phorbol myristic acetate. On Sephadex G75, the T lymphocyte (or T lymphocyte supernatants) induced TPF eluted as a single peak with a molecular weight of approximately 16,000 Daltons. Chromatography on DEAE cellulose resulted in the fractionation of TPF into at least four peaks of activity. The TPF generated in this system was shown to be P388D1-derived and thus distinct from lymphocyte-derived mitogenic factor. However, TPF appears to be functionally, and in part, chemically similar to the lymphocyte activating factor (LAF) described by Gery et al. (J. Immunol. 136: 128, 1972). Thus, the P388D1 cell line will be a very useful tool for the isolation and characterization of macrophage-derived factors which are mitogenic for thymocytes.